This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The kinetochores is a multi-protein complex that assemblies on the centromere and attaches chromosomes to spindle microtubules during cell division. Its attachment to chromosomes is tightly monitored to ensure faithful chromosome segregation. A recent systematic yeast two-hybrid screen focusing on mitotic spindle function identified interactions between the kinetochore protein Mif2 and two Casein Kinase 2 (CK2) subunits, Cka2 and Ckb2. CK2 is a ubiquitous and highly conserved Ser/Thr kinase in eukaryotes. Although CK2 has been linked to cell cycle regulation, it has remained largely unclear how CK2 regulates this event. Additionally, Mif2 and another kinetochore protein Ndc10 (Cbf2) were previously identified as putative substrates of CK2. Ndc10 is a component of the CBF3 complex. Mif2 is the S. cerevisiae homolog of human CENP-C. Both proteins bind to centromeres and recruit other inner and outer kinetochore proteins. Previous studies have demonstrated that both Mif2 and Ndc10 are phosphorylated by Ipl1, the yeast Aurora B kinase. However, the role of phosphorylation has not been fully explored. The overall goal of my project is to determine how CK2 regulates kinetochore function. I hypothesize that CK2 regulates mitotic progression by regulating Aurora B phosphorylation of Mif2 and Ndc10. To test these hypotheses, I will first determine if loss of Cka2 kinase activity affects mitotic spindle and chromosome segregation. Genetic interaction between cka2-ts and ipl1-ts will be examined. In addition to cka2- ts mutants, cka2-as (analogue sensitive) mutants will be created and tested. I will also test if Mif2 andNdc10 are phosphorylated by CK2 in vitro and in vivo, and their phosphorylation sites will be mapped by mass spectrometry. Both Mif2 and Ndc10 have multiple putative Ipl1 phosphorylation sites. How phosphorylation by one kinase affects phosphorylation by the other will be investigated in vitro and in vivo. Moreover, phosphorylation sites of Mif2 and Ndc10 will be mutagenized and the role of these modifications will be examined. Finally, using cka2-as mutants, a screen focusing on kinetochores and their regulatory proteins will be conducted to identify additionalCK2 substrates. Understanding how CK2 regulates kinetochores will provide novel insights into the role of CK2 in cell cycle regulation.